The guanine nucleotide exchange factor (GEF) from rabbit reticulocytes which consists of five subunits will be further purified (Proc. Natl. Acad. Sci. U.S.A. 83, 6746, (1986)) in order to study its interaction with eukaryotic chain initiation factor 2 (eIF-2). By using fluorescent probes covalently attached to eIF- 2, the molecular interaction of eIF-2, GEF and nucleotides will be monitored. The intrinsic fluorescence of GEF-bound NADPH will also be used as a probe for the interaction of GEF with eIF-2, GTP, GDP and GTP analogs and Met-tRNAf. The role of NADP/NADPH in regulating nucleotide exchange and its effect on protein synthesis will be examined. We will also determine which subunits of elF-2 and GEF are responsible for protein: protein interaction and nucleotide binding. The phosphorylation of GEF under conditions of the nucleotide exchange reaction and its effect on GEF activity will be studied. The binding of the eukaryotic polypeptide chain initiation factors 4A, 4B, and 4F to poly(l,N6 -ethenoadenylic acid) will be investigated by fluorescence spectroscopy. Competition experiments will allow us to determine the relative affinity of these proteins for mRNA cap analogs and different triplets. The effect of ATP, Mg2+, and polyamines on these reactions will be determined. We will also label eIF-4F with fluorescent probes in order to monitor the cooperative interactions of eIF-4F, messenger RNA and eIF-4B. The role of eIF-4D and Ca2+ in protein synthesis will be investigated. We will quantitate the binding of Ca2+ to eIF-4D. We will also examine the activation of transglutaminase by elF-4D and calmodulin. A mouse Friend erythroleukemic cell lysate will be utilized to evaluate the roles of elF-4D, Ca2+, and calmodulin in regulating protein synthesis. An Artemia cDNA library which was constructed in the lambda gtll expression vector will be screened with synthetic oligonucleotide probes and antibodies specific for the alpha and beta subunits of Artemia elF-2. The isolated clones will be characterized by restriction mapping, nucleotide sequencing, and their derived amino acid sequence. The sequencing of the genomic clones for